Molecular torches and their use under denaturing conditions

ABSTRACT

The present invention features “molecular torches” and the use of molecular torches for detecting the presence of a target nucleic acid sequence. Molecular torches contain a target binding domain, a target closing domain, and a joining region. The target binding domain is biased towards the target sequence such that the target binding domain forms a more stable hybrid with the target sequence than with the target closing domain under the same hybridization conditions. The joining region facilitates the formation or maintenance of a closed torch.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/352,355, filed Jan. 27, 2003, now pending, which is a continuation ofU.S. application Ser. No. 10/001,344, filed Oct. 31, 2001, now U.S. Pat.No. 6,534,274, which is a continuation of U.S. application Ser. No.09/346,551, filed Jul. 1, 1999, now U.S. Pat. No. 6,361,945, whichclaims the benefit of U.S. Provisional Application No. 60/091,616, filedJul. 2, 1998. The contents of each of the foregoing applications ishereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for detectingthe presence or amount of a target nucleic acid sequence in a sample.

BACKGROUND OF THE INVENTION

None of the references described herein are admitted to be prior art tothe claimed invention.

A target nucleic acid sequence can be detected by various methods usingnucleic acid probes designed to preferentially hybridize to the targetsequence over other sequences that may be present in a sample. Examplesof target sequences include sequences that may be initially present in asample, or produced as part of an amplification procedure, such as asequence characteristic of a microorganism, a virus, a plant gene, or ananimal gene such as a human gene. A reporter sequence which is producedas part of a detection method in the presence of a target sequence, butwhich has a sequence that is not dependent on the target sequence, canalso be detected.

Hybridization of probes to target nucleic acid sequences can formdetectable probe:target duplexes under appropriate conditions. Detectionof such duplexes is facilitated using a labeled probe. Differenttechniques are available to reduce background due to signal from labeledprobes not hybridized to a target sequence. Such techniques includeusing a physical separation step, a label preferentially altered in aprobe:target duplex versus an unhybridized probe, and/or interactinglabels.

Interacting labels are two or more labels which cooperate when in closeproximity to one another to produce a signal which is different from asignal produced from such labels when they are farther apart so thattheir cooperation is diminished. The labels may be associated with oneor more molecular entities. Detection systems can be designed such thatthe labels interact either in the presence of a target sequence or inthe absence of a target sequence.

Taub et al., U.S. Pat. No. 4,820,630 describes interacting labelspresent on two different nucleic acid molecules cooperating to produce adetectable signal in the presence of a target nucleic acid sequence.Hybridization of both molecules to the target sequence brings the labelsinto close proximity so that they can cooperate to produce a signaldifferent from labels not cooperating in close proximity.

Morrison, European Application Number 87300195.2, Publication Number 0232 967, describes a detection system involving a reagent made up of twocomplementary nucleic acid probes. One of the complementary probescontains a first label, and the other complementary probe contains asecond label. The first and the second labels can interact with eachother. Formation of a complex between the target sequence and one of thetwo complementary probes changes the interaction between the two labels.

Lizardi et al., U.S. Pat. Nos. 5,118,801 and 5,312,728, describes anucleic acid probe containing a target complementary sequence flanked by“switch” sequences that are complementary to each other. In the absenceof a target sequence, the switch sequences are hybridized together. Inthe presence of a target sequence the probe hybridizes to the targetsequence, mechanically separating the switch sequences and therebyproducing an “open switch”. The state of the switch sequence, whetheropen or closed, is indicated to be useful for selectively generating adetectable signal if the probe is hybridized to the target sequence.

Lizardi et al., International Application Number PCT/US94/13415,International Publication WO 95/13399, describes a “unitary”hybridization probe. The probe contains a target complementary sequence,an affinity pair holding the probe in a closed conformation in theabsence of target sequence, and a label pair that interacts when theprobe is in a closed conformation. Hybridization of the probe to thetarget sequence shifts the probe to an open conformation, which reducesthe interaction between the label pair.

SUMMARY OF THE INVENTION

The present invention features “molecular torches” and the use ofmolecular torches for detecting the presence of a target nucleic acidsequence. Molecular torches contain a target binding domain, a targetclosing domain, and a joining region. The target binding domain isbiased towards the target sequence such that the target binding domainforms a more stable hybrid with the target sequence than with the targetclosing domain under the same hybridization conditions. The joiningregion facilitates the formation or maintenance of a closed torch.

The presence of a target sequence can be detected using a moleculartorch by measuring whether the molecular torch is opened or closed. In a“closed torch” the target binding domain is hybridized to the targetclosing domain. In an “open torch” the target binding domain is nothybridized to the target closing domain.

The target sequence bias of the molecular torch target binding domain,and the joining region, are preferably used to detect a target sequencein conjunction with (1) target binding domain denaturing conditions andtarget binding domain hybridizing conditions, or (2) strand displacementconditions.

Under target binding domain denaturing conditions the torch is open andreadily accessible for hybridization to the target sequence. The targetbinding domain bias towards the target sequence allows the targetbinding domain to remain open in the presence of target sequence due tothe formation of a target binding domain:target sequence hybrid evenwhen the sample stringency conditions are lowered.

Under strand displacement conditions the target sequence can hybridizewith the target binding domain present in a closed torch to thereby openthe torch. Assays carried out using strand displacement conditions canbe preformed under essentially constant environmental conditions. Underessentially constant environmental conditions the environment is notchanged to first achieve denaturation and then achieve hybridization,for example, by raising and lowering the temperature.

The joining region facilitates the production or maintenance of a closedtorch by producing at least one of the following: (1) an increase in therate of formation of the closed torch; and (2) an increase in thestability of the closed torch. The increase in the rate of formationand/or stability is with respect to such activities in the absence of ajoining region.

The joining region is made up of one or more groups that covalentlyand/or non-covalently link the target opening and target closing domainstogether. Individual groups present in the joining region are joinedtogether by covalent and/or non-covalent interactions such as ionicinteraction, hydrophobic interaction, and hydrogen bonding.

Detecting the presence of an open torch includes directly detectingwhether open torches are present and/or detecting whether closed torchesare present. Examples of techniques that can be used to detect opentorches include the following: (1) those involving the use ofinteracting labels to produce different signals depending upon whetherthe torch is open or closed; (2) those involving the use of a targetclosing domain comprising a label that produces a signal when in atarget binding domain:target closing domain hybrid that is differentthan the signal produced when the target closing domain is nothybridized to the target binding domain; and (3) those involving thedetection of sequence information made available by an open targetbinding domain.

Preferably, interacting labels are used for detecting the presence of anopen torch. Techniques involving the use of interacting labels can becarried out using labels that produce a different signal when they arepositioned in close proximity to each other due to a closed targetbinding domain than when they are not in close proximity to each otheras in an open target binding domain. Examples of interacting labelsinclude enzyme/substrates, enzyme/cofactor, luminescent/quencher,luminescent/adduct, dye dimers, and Forrester energy transfer pairs.

The target binding domain and the target closing domains are made up ofnucleotide base recognition sequences that are substantiallycomplementary to each other. A “nucleotide base recognition sequence”refers to nucleotide base recognition groups covalently linked togetherby a backbone. Nucleotide base recognition groups can hydrogen bond, atleast, to adenine, guanine, cytosine, thymine or uracil. A nucleotidebase recognition sequence “backbone” is made up of one or more groupscovalently joined together that provide the nucleotide base recognitiongroups with the proper orientation to allow for hybridization tocomplementary nucleotides present on nucleic acid.

“Substantially complementary sequences” are two nucleotide baserecognition sequences able to form a stable hybrid under conditionsemployed. Substantially complementary sequences may be present on thesame or on different molecules.

Substantially complementary sequences include sequences fullycomplementary to each other, and sequences of lesser complementarity,including those with mismatches and with linkers. Bugles, such as thosedue to internal non-complementary nucleotides, and non-nucleotidelinkers, placed between two recognition groups hybridized together mayalso be present. Preferably, substantially complementary sequences aremade up of two sequences containing regions that are preferably at least10, at least 15, or at least 20 groups in length. Preferably, at least70%, at least 80%, at least 90%, or 100% of the groups present in one ofthe two regions hydrogen bond with groups present on the other of thetwo regions. More preferably, hydrogen bonding is between complementarynucleotide bases A-T, G-C, or A-U.

A “linker” refers to a chain of atoms covalently joining together twogroups. The chain of atoms are covalently joined together and caninclude different structures such as branches and cyclic groups.

Thus, a first aspect of the present invention features the use of amolecular torch to determine whether a target nucleic acid sequence ispresent in a sample. The molecular torch comprises: (1) a targetdetection means for hybridizing to the target sequence, if present, toproduce an open torch; (2) torch closing means for hybridizing to thetarget detecting means in the absence of the target sequence to providea closed torch conformation; and (3) joining means joining the targetdetection means and the torch closing means. Detecting the presence ofthe open torch provides an indication of the presence of the targetsequence.

“Target detection means” refers to material described in the presentapplication and equivalents thereof that can hybridize to the targetsequence and the torch closing means. The target detection means isbiased toward the target sequence, as compared to the torch closingmeans, such that in the presence of the target sequence the targetdetection means preferentially hybridizes to the target sequence.

“Torch closing means” refers to material described in the presentapplication and equivalents thereof that can hybridize to the targetdetection means to provide a closed torch.

“Joining means” refers to material described in the present applicationand equivalents thereof that join the target detection means and thetorch closing means, and that facilitate the production or maintenanceof a closed torch in the absence of a target sequence.

Another aspect of the present invention features the use of a moleculartorch to determine whether a target sequence is present involving thefollowing steps: (a) contacting a sample with a molecular torchcontaining a target binding domain and a target closing domain connectedtogether by a joining region; and (b) detecting the presence of an opentorch as an indication of the presence of the target sequence.

The target binding domain is biased towards the target sequence suchthat a target binding domain:target sequence hybrid is more stable thana target binding domain:target closing domain hybrid. If the targetsequence is not present, the closed torch conformation is favored.

Before being exposed to the sample, the molecular torch target bindingdomain may be open or closed depending upon the environment where it iskept. Denaturing conditions can be used to open up the target bindingdomain. Preferably, denaturation is achieved using heat.

Alternatively, strand displacement conditions can be employed. If stranddisplacement conditions are employed, then the molecular torch does notneed to be opened before binding the target sequence.

Another aspect of the present invention describes a method of detectingthe presence of a target sequence where a mixture containing a sampleand a molecular torch is first exposed to denaturing conditions and thenexposed to hybridization conditions. The presence of an open torch isused an indication of the presence of the target sequence.

“Denaturing conditions” are conditions under which the target bindingdomain:target closing domain hybrid is not stable and the torch is open.In a preferred embodiment, the joining region remains intact under thedenaturing conditions. Thus, in this preferred embodiment, underdenaturing conditions the target binding domain becomes available forhybridization to the target sequence, but is also kept in proximity tothe target closing domain for subsequent hybridization in the absence ofthe target sequence.

“Hybridization conditions” are conditions under which both the targetbinding domain:target closing domain hybrid and the target bindingdomain:target sequence hybrid are stable. Under such conditions, in theabsence of the target sequence, the target binding domain is notinhibited by hybridized target sequence from being present in a hybridwith the target closing domain.

Another aspect of the present invention describes a molecular torch. Themolecular torch contains (1) a target detection means for hybridizing toa target sequence, if present, to produce an open torch; (2) a torchclosing means for hybridizing to the target detecting means in theabsence of the target sequence to provide a closed torch; and (3) ajoining means for facilitating a closed torch conformation in theabsence of the target sequence.

Another aspect of the present invention describes a molecular torchcontaining a target binding domain and a target closing domain joinedtogether through a joining region. The target binding and target closingdomains are substantially complementary to each other. The targetbinding domain is biased to a target sequence that is a perfect DNA orRNA complement, preferably RNA complement, of the target binding domain.Thus, the target binding domain forms a more stable duplex with itsprefect DNA or RNA complement than with the target closing domain.

A “perfect DNA or RNA complement of the target binding domain” is a DNAor RNA containing a complementary purine or pyrimidine nucleotide baseopposite each recognition group present in the target binding domain.The complementary purine or pyrimidine nucleotide bases can hydrogenbond to each other.

Various examples are described herein. These examples are not intendedin any way to limit the claimed invention.

Other features and advantages of the invention will be apparent from thefollowing drawing, the description of the invention, the examples, andthe claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides an energy diagram illustrating the free energy of atarget binding domain:target closing domain hybrid (I), the stability ofa target binding domain:target sequence hybrid (II), the difference inthe free energy of I and II (ΔG), and the difference in the activationfree energy (ΔG*) for the conversion of I into II.

FIGS. 2A-2G provides examples of different molecular torch structures.“F” refers to fluorophore and “Q” refers to quencher.

FIGS. 3A-3C provide examples of strand displacement. “F” refers tofluorophore and “Q” refers to quencher. Target sequence is shown by abolded line.

FIG. 4 illustrates the functioning of a molecular torch containing ajoining region that is a covalent linkage. “F” refers to fluorophore and“Q” refers to quencher.

FIG. 5 illustrates the functioning of a molecular torch containing ajoining region made up of two polyethylene glycol (PEG) groups and twosubstantially complementary nucleic acid sequences having a sufficientlyhigh T_(m) so as not to melt during heating. “F” refers to fluorophoreand “Q” refers to quencher.

FIGS. 6A and 6B illustrate strands making up molecular torches 1-7. “F”refers to a fluorophore, “Q” refers to a quencher, “PEG” refers topolyethylene glycol, and “ccc” refers to a propyl group located at the3′-position of the terminal sugar. Bases shown in italics are 2′-methoxysubstituted ribonucleotides.

FIG. 7 illustrates a molecular torch which can be used in a stranddisplacement reaction. “F” refers to fluorophore and “Q” refers toquencher.

FIG. 8 illustrates the functioning of a molecular torch in a stranddisplacement reaction. “F” refers to fluorphore and “Q” refers toquencher. Target sequence is shown by a bolded line.

DETAILED DESCRIPTION OF THE INVENTION

A molecular torch is preferably designed to provide favorable kineticand thermodynamic components in an assay to detect the presence of atarget sequence. The kinetic and thermodynamic components of an assayinvolving a molecular torch can be used to enhance the specificdetection of a target sequence.

The thermodynamics of a preferred molecular torch are illustrated inFIG. 1. Referring to FIG. 1, “I” denotes the target bindingdomain:target closing domain hybrid while “II” denotes the targetbinding domain:target sequence hybrid. In FIG. 1, ΔG* represents thefree energy of activation required to melt the target bindingdomain:target closing domain, and ΔG represents the difference in freeenergy between the target binding domain:target closing domain hybridand the target binding domain:target sequence hybrid.

The thermodynamic component of the present invention is based upon thetarget binding domain:target sequence hybrid being more stable than thetarget binding domain:target closing domain hybrid (ΔG<0). The joiningregion facilitates the closed torch conformation in the absence of thetarget sequence.

Additionally, depending upon the torch design, the joining region can beused to provide one or more of the following advantages: (1) reducingthe probability of labels present on the target opening and closingdomains coming apart in the absence of target; (2) facilitating the useof short target binding domains which can be used to enhance itssensitivity to mismatched targets; (3) facilitating a closed torchconformation when the target closing domain and the target bindingdomain contains, for example, mismatches or abasic “nucleotides”; and(4) facilitating the detection of adenine and thymine rich targetsequences by stabilizing interaction of adenine and thymine rich targetbinding and target closing domains.

Denaturing Conditions

Denaturing conditions can be used to provide sufficient energy (ΔG*) tomelt the target binding domain:target closing domain hybrid. The amountof energy required will vary depending upon the molecular torchcomposition and the environmental conditions. The environmentalconditions include the assay solution composition and temperature. Thenecessary energy needed to open a closed target can be supplied, forexample, by heating the sample.

A useful measure of the stability of a hybrid is the melting temperature(T_(m)). At the melting temperature 50% of the hybrids present aredenatured.

Using a particular assay composition, a hybrid is not stable when theassay temperature is above the T_(m). Depending upon the composition ofan assay, the T_(m) of a hybrid will vary. Factors such as saltconcentration and the presence of denaturing agents can affect the T_(m)of a given hybrid. The T_(m) is determined using a particular assaycomposition and varying the temperature.

By taking into account factors affecting T_(m), such as those describedherein and those well known in the art, molecular torches can be readilydesigned to have a desirable target binding domain:target closing domainT_(m) and a desirable target binding domain:target T_(m) such that ΔG<0.The T_(m) can be measured, for example, using techniques such as thosedescribed by Sambrook et al., Molecular Cloning a Laboratory Manual,Second ed., Cold Spring Harbor Laboratory Press, 1989, and Hogan et al.,U.S. Pat. No. 5,547,842 (both of which are hereby incorporated byreference herein).

While FIG. 2 shows a number of different molecular torch configurations,those skilled in the art will readily appreciate other molecular torchconfigurations which may be used in practicing the present invention.FIG. 2A illustrates a two-stranded molecular torch made up of a targetbinding region and a joining region. The target binding region consistsof a target binding domain that binds the target sequence and a targetclosing domain that binds the target binding domain.

FIGS. 2B-2D illustrate single-stranded molecular torches composed oftarget binding and joining regions, while FIG. 2E illustrates athree-stranded molecular torch.

FIG. 2F illustrates a molecular torch containing a joining region andtwo target binding regions. The two target binding regions can bind thesame or different target sequences and can have the same or differentinteracting labels. For example, by positioning different types offluorophores having different emission characteristics that areseparately detectable on each target binding region the presence ofdifferent target sequences can be detected using a single moleculartorch by looking for the signal characteristics of the differentfluorophores.

FIG. 2G illustrates a two-stranded molecular torch made up of a targetbinding region and a joining region. The joining region containscomplementary polynucleotides joined to the target binding or targetclosing domains by a linker made up, for example, of PEG or apolynucleotide.

Strand Displacement Conditions

Under strand displacement conditions the target binding domain:targetsequence hybrid is more stable than the target binding domain:targetclosing domain hybrid, and production of the target bindingdomain:target sequence hybrid is favored if the target sequence ispresent.

Strand displacement is preferably performed using torches havingnucleotide base recognition groups accessible for hybridization totarget. Such torches preferably contain one to about ten nucleotide baserecognition groups complementary to the target sequence which areaccessible. Preferably, no more than ten, five or three nucleotide baserecognition groups are accessible.

Different configurations are possible, including those where thesingle-stranded region is a terminal region, or where thesingle-stranded region is an internal region such as a loop region.Alternatively, strand displacement conditions causing, for example, the5′ or 3′ terminal torch region to “breath” may be employed. Breathing ofa torch occurs under conditions where the stability of a region allowsthe torch to become single-stranded and hybridize to the target sequencesuch that formation of the target binding domain:target sequence hybridis favored.

FIGS. 3A-3C provides different examples of strand displacement. FIGS. 3Aand 3C illustrate molecular torches having three terminal nucleotidesavailable for target hybridization. FIG. 3B illustrates breathing of twoterminal nucleotides and target hybridization.

Target Sequence Bias

The target binding domain can be biased towards the target sequenceusing different design considerations affecting nucleic acid hybridstability. Such considerations include the degree of complementarity,the type of complementary recognition groups, and the nucleotide baserecognition sequence backbone. The affect of these different factorsvaries depending upon the environmental conditions.

The degree of complementarity takes into account the number ofrecognition groups present on the target binding domain that hydrogenbond with recognition groups present on the target closing domain andwith the target sequence. The target binding domain can be designed tohave a greater degree of complementarity to the target sequence than tothe target closing domain using different techniques. Such techniquesinclude, for example, designing the target binding domain to havemismatches with the target closing domain but not the target sequenceand the use of non-nucleotide linkers in the target closing domain.

Examples of non-nucleotide linkers present in a nucleotide baserecognition sequence are abasic “nucleotides”. Abasic “nucleotides” lacka nucleotide base recognition group.

Other examples of non-nucleotide linkers include polysaccharides,peptides, and polypeptides. Arnold et al. International Application No.PCT/US88/03173, International Publication WO 89/02439, and U.S. Pat. No.5,585,481, hereby incorporated by reference herein, also provideexamples of non-nucleotide linkers.

The types of recognition groups present can be used to bias the targetbinding domain towards the target sequence by taking into accountfactors such as the degree of hydrogen bonding between differentnucleotide purine and pyrimidine bases. For example, G-C pairing or 2,6diaminopurine-thymine is stronger than A-T pairing and pairing withuniversal bases such as inosine. The target binding domain can bedesigned to have increased G or C pairing with nucleotides present in atarget sequence compared to the target closing domain.

The composition of nucleotide base recognition sequence backbones can beadjusted in different ways to bias the target binding domain towards atarget sequence. Preferred molecular torch backbones aresugar-phosphodiester type linkages, such as those present in ribo- anddeoxyribonucleic acids. Another type of linkage is a peptide linkage,such as that present in peptide nucleic acids.

Peptide nucleic acids generally form a more stable duplex with RNA thanwith the corresponding DNA sequence. Thus, the target binding domain canbe biased towards an RNA target sequence, for example, by using amolecular torch where the target binding domain contains peptide nucleicacid groups and the target closing domain is made up of DNA.

In the case of a sugar-phosphodiester type linkage, both the sugar groupand the linkage joining two sugar groups will affect hybrid stability.An example of the affect the sugar can have is that seen with 2′-methoxysubstituted RNA. 2′-methoxy containing nucleic acids generally form morestable duplexes with RNA than with the corresponding DNA sequence.Another example, is 2′-fluoro substituted RNA that has the same type ofaffect as 2′-methoxy substituted RNA.

Examples of ways in which the backbone linking group may affect hybridstability include affecting the charge density and the physicalassociation between two strands. Steric interactions from bulky groupscan reduce hybrid stability. Groups such as phosphorothioates can reducehybrid stability, whereas uncharged groups such as methylphosphonatescan increase hybrid stability.

Target Binding Domain:Target Sequence Hybrid

Formation of a target binding domain:target sequence hybrid results inthe production of an open torch that is more stable than a closed torch.Conditions for opening up of the torch, or strand displacement, can beused to facilitate the production of an open torch in the presence of atarget sequence.

Opening and closing of the torch can be achieved by changing theenvironmental conditions of the detection method employed. Examples ofchanges to the environmental conditions to open and close the torchinclude heating and cooling; raising and lowering the pH; and adding adenaturing agent, then diluting out the agent.

The target binding domain:target sequence hybrid is more stable than thetarget binding domain:target closing domain hybrid. Preferably, underconditions used in the detection method, the target bindingdomain:target T_(m) is at least 2° C., more preferably at least 5° C.,even more preferably at least 10° C., more than the target bindingdomain:target closing domain T_(m).

A closed torch in the absence of a target sequence reduces backgroundfrom a molecular torch not hybridized to the target sequence without theneed for a separation step. Preferably, in those assays where the torchis first opened, hybridization conditions closing the torch in theabsence of a target sequence employ a temperature that is at least 2° C.lower, more preferably at least 5° C. lower, and more preferably atleast 110° C. lower, than the T_(m) of the target binding domain:targetclosing domain hybrid.

If desired, a separation step can be employed to physically separatemolecular torches hybridized to target sequences from molecular torchesnot hybridized to target sequences. A separation step can be carriedout, for example, using sequence information made available by the opentarget binding domain. For example, a capture probe having a nucleicacid sequence complementary to the target closing domain can be used tocapture a molecular torch hybridized to a target sequence. The captureprobe itself may be provided either directly or indirectly on a bead orcolumn.

If capture probes, or other types of nucleic acid probes complementaryto the target closing domain are used, it is important that such probesbe designed and used under conditions where a stable target closingdomain:probe hybrid is not formed in the absence of a target bindingdomain:target sequence hybrid. Preferably, a target closing domain:probehybrid has a T_(m) that is at least 5° C., and more preferably at least10° C. lower than a target binding domain:target closing domain hybrid.

Detecting the Target Sequence

Molecular torches can be used to detect the presence of a targetsequence by determining whether the torch is open under conditions wherea target binding domain:target closing domain hybrid is stable. Opentorches can be detected using different techniques such as (1) thoseinvolving the use of interacting labels to produce different signalsdepending upon whether the torch is open or closed; (2) those involvingthe use of a target closing domain comprising a label that produces asignal when in a target binding domain:target closing domain hybrid thatis different from the signal produced when the target closing domain isnot hybridized to the target binding domain; and (3) those involving thedetection of sequence information made available by an open targetbinding domain.

Different types of interacting labels can be used to determine whether atorch is open. Preferably, the interacting labels are either aluminescent/quencher pair, luminescent/adduct pair, Forrester energytransfer pair or a dye dimer. More than one label, and more than onetype of label, may be present on a particular molecule.

A luminescent/quencher pair is made up of one or more luminescentlabels, such as chemiluminescent or fluorescent labels, and one or morequenchers. Preferably, a fluorescent/quencher pair is used to detect anopen torch. A fluorescent label absorbs light of a particularwavelength, or wavelength range, and emits light with a particularemission wavelength, or wavelength range. A quencher dampens, partiallyor completely, signal emitted from an excited fluorescent label.Quenchers can dampen signal production from different fluorophores. Forexample, 4-(4′-dimethyl-amino-phenylaxo)benzoic acid (DABCYL) can quenchabout 95% of the signal produced from5-(2′-aminoethyl)aminoaphthaline-1-sulfonic acid (EDANS), rhodamine andfluorescein.

Different numbers and types of fluorescent and quencher labels can beused. For example, multiple fluorescent labels can be used to increasesignal production from an opened torch, and multiple quenchers can beused to help ensure that in the absence of a target sequence an excitedfluorescent molecule produces little or no signal. Examples offluorophores include acridine, fluorescein, sulforhodamine 101,rhodamine, EDANS, Texas Red, Eosine, Bodipy and lucifer yellow. (E.g.,see Tyagi et al., Nature Biotechnology 16:49-53, 1998, herebyincorporated by reference herein). Examples of quenchers include DABCYL,Thallium, Cesium, and p-xylene-bis-pyridinium bromide.

A luminescent/adduct pair is made up of one or more luminescent labelsand one or more molecules able to form an adduct with the luminescentmolecule(s) and, thereby, diminish signal production from theluminescent molecule(s). The use of adduct formation to alter signalsfrom a luminescent molecule using ligands free in solution is describedby Becker and Nelson, U.S. Pat. No. 5,731,148, hereby incorporated byreference herein. Adducts can also be formed by attaching an adductformer to the molecular torch, or to a nucleic acid probe thathybridizes with sequence information made available in an open torch.

Forrester energy transfer pairs are made up of two labels where theemission spectra of a first label overlaps with the excitation spectraof a second label. The first label can be excited and emissioncharacteristic of the second label can be measured to determine if thelabels are interacting. Examples of Forrester energy transfer pairsinclude pairs involving fluorescein and rhodamine;nitrobenz-2-oxa-1,3-diazole and rhodamine;

-   -   fluorescein and tetramethylrhodamine; fluorescein and        fluorescein; IAEDANS and fluorescein; and BODIPYFL and        BIODIPYFL.

A dye dimer is made up of two dyes that interact upon formation of adimer to produce a different signal than when the dyes are not in adimer conformation. Dye dimer interactions are described, for example,by Packard et al., Proc. Natl. Sci. USA 93:11640-11645, 1996 (which ishereby incorporated by reference herein).

The observed signal produced during the detection step that ischaracteristic of the presence of a target sequence can be comparedagainst a control reaction having no target sequence or known amounts oftarget sequences. Known amounts of target sequences can be used toobtain a calibration curve. While a control reaction is preferablyperformed at the same time as an experimental reaction, controlreactions do not need to be run at the same time as the experimentalreaction and can be based on data obtained from a previous experiment.

Examples of using molecular torches having interacting labels to detecta target sequence are provided in FIGS. 4 and 5. Both figures illustratethe presence of a target sequence. In the absence of the targetsequence, the molecular torch target binding domain is closed resultingin the quenching of signal.

FIG. 4 illustrates the use of a single-stranded molecular torchcontaining a small joining region. Heat is used to melt the targetbinding domain:target closing domain hybrid. The torch is biased towardsan RNA target sequence by, for example, the presence of 2′-methoxysubstituted ribonucleotides present in the target binding domain. In thepresence of the target sequence the quencher (O) is no longer held inclose proximity to the fluorophore (F), thus, decreasing the ability ofthe quencher to affect fluorophore fluorescence.

FIG. 5 illustrates the use of a double-stranded molecule with a joiningregion made up of two parts, a non-nucleotide PEG linker and a sequencewhose high T_(m) prevents its melting during the assay.

Another embodiment of the present invention involves detecting opentorches using a label producing a signal when in a target bindingdomain:target closing domain hybrid that is different from the signalproduced when the target closing domain is not hybridized to the targetbinding domain. Such labels include luminescent molecules and labelshaving a different stability when present in different environments.

Signal produced from luminescent molecules present on one nucleotidebase recognition sequence can be affected by another nucleotide baserecognition sequence. For example, nucleotides on one nucleotide baserecognition sequence can be used to quench, or effect the rotationalmotion, of a fluorophore present on another nucleotide base recognitionsequence.

Environments that can affect the stability of certain labels include anucleic acid duplex formed with Watson-Crick base pairing. Examples ofsuch labels and there use are described by Becker and Nelson, U.S. Pat.No. 5,731,148, and Arnold et al., U.S. Pat. No. 5,283,174, both of whichare hereby incorporated by reference herein.

Acridinium ester and derivatives thereof are preferred examples oflabels for detecting open torches based on the environment of the label.An acridinium ester can be detected using different techniques such asselectively inactivating label not present in a nucleic acid duplex. Anexample of the use of one or more acridinium ester labels involvesattaching such labels to the target closing domain and using a reductionin signal due to selective inactivation of acridinium ester label(s)present on a single-stranded target closing domain as an indication ofthe presence of target sequence.

The detection of open torches using sequence information made availableby an open target binding domain can be carried out using detectionprobes that hybridize to the target closing domain. Preferred detectionprobes contain a detectable label. The detectable label can, forexample, be a label interacting with a label present on the targetclosing domain, or can be a label that produces a signal in the absenceof an interacting label on the target closing domain. A preferred probelabel is an acridinium ester.

Increasing the Number of Target Sequences

In cases where a target sequence is present in a sample in low numbers,an amplification can be performed to increase the number of targetsequences. Numerous amplification techniques are well known in the artincluding those involving transcription-associated amplification, thepolymerase chain reaction (PCR) and the ligase chain reaction (LCR).

Preferably, the molecular torch is used in conjunction with atranscription-associated amplification. Transcription-associatedamplification involves generating RNA transcripts using an RNApolymerase that recognizes a double-stranded DNA promoter region.

Examples of references describing transcription-associated amplificationinclude Burg et al., U.S. Pat. No. 5,437,990; Kacian et al., U.S. Pat.No. 5,399,491; Kacian et al., U.S. Pat. No. 5,554,516; Kacian et al.,International Application No. PCT/US93/04015, International PublicationWO 93/22461; Gingeras et al., International Application No.PCT/US87/01966, International Publication WO 88/01302; Gingeras et al.,International Application No. PCT/US88/02108, International PublicationWO 88/10315; Davey and Malek, EPO Application No. 88113948.9, EuropeanPublication No. 0 329 822 A2; Malek et al., U.S. Pat. No. 5,130,238;Urdea, International Application No. PCT/US91/00213, InternationalPublication WO 91/10746; McDonough et al., International Application No.PCT/US93/07138, International Publication WO 94/03472; and Ryder et al.,International Application No. PCT/US94/08307, International PublicationWO 95/03430. (Each of these references is hereby incorporated byreference herein.)

The use of a transcription-associated amplification procedure involvingRNase H activity is preferred. More preferably, the procedure utilizesRNase H activity present in reverse transcriptase to facilitate strandseparation. Kacian et al., U.S. Pat. No. 5,399,491 describes anamplification occurring under essentially constant conditions withoutthe addition of exogenous RNase H activity. The procedure utilizes RNaseH activity present in reverse transcriptase to facilitate strandseparation.

One of the advantages of using the present invention in conjunction witha transcription-associated amplification is that the molecular torch canbe added prior to amplification, and detection can be carried outwithout adding additional reagents. The molecular torch is well suitedfor use in a transcription-associated amplification because the T_(m) ofthe target binding domain:target closing domain hybrid can readily beadjusted to be higher than the temperature used during theamplification. The closed target binding domain prevents the moleculartorch from prematurely binding to target sequences generated byamplification.

After amplification, the solution can be heated to open the targetbinding domain allowing the molecular torch to hybridize to a targetsequence. The solution can then be cooled to close target bindingdomains of torches not hybridized to target sequences. The presence ofopen torches having, for example, a fluorophore/quencher pair can thenbe measured by irradiating the sample with the appropriate excitationlight and then measuring emission light.

Examples of references mentioning other amplification methods includethose describing PCR amplification such as Mullis et al., U.S. Pat. Nos.4,683,195, 4,683,202, and 4,800,159, and Methods in Enzymology, Volume155, 1987, pp. 335-350; and those describing the ligase chain reaction,such as Backman, European Patent Application No. 88311741.8, EuropeanPublication No. 0 320 308. (Each of these references is herebyincorporated by reference herein.)

Molecular Torch Construction

A molecular torch comprises a target binding domain, a target closingdomain and a joining region. The target binding and closing domains areeach nucleotide base recognition groups.

Nucleotide base recognition sequences contain nucleotide baserecognition groups able to hydrogen bond with nucleotide nitrogenousbases present in nucleic acid. The nucleotide base recognition groupsare joined together by a backbone providing a proper conformation andspacing to allow the groups to hydrogen bond to nucleotides present onnucleic acid.

A given nucleotide base recognition group may be complementary to aparticular nucleotide (e.g., adenine, guanine, cytosine, thymine, anduracil) and, thus, be able to hydrogen bond with that nucleotide presentin a nucleic acid. A nucleotide base recognition group may also be ableto hydrogen bond with different nucleotides. For example, when inosineis a nucleotide base recognition group it can hydrogen bond withdifferent nucleotide bases.

Preferred nucleotide base recognition groups are nitrogenous purine orpyrimidine bases, or derivatives thereof, able to hydrogen bond witheither adenine, guanine, cytosine, thymine or uracil. Examples of suchrecognition groups include adenine, guanine, cytosine, thymine, uracil,and derivatives thereof. Examples of derivatives include modified purineor pyrimidine bases such as N4-methyl deoxyguanosine, deaza or azapurines and pyrimidines used in place of natural purine and pyrimidinebases, pyrimidine bases having substituent groups at the 5 or 6position, and purine bases having an altered or a replacementsubstituent at the 2, 6 or 8 position. See, e.g., Cook, InternationalApplication No. PCT/US92/11339, International Publication WO 93/13121(hereby incorporated by reference herein). Additional examples include,2-amino-6-methylaminopurine, 06-methylguanine, 4-thio-pyrimidines,4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines andO4-alkyl-pyrimidines (see, e.g., The Glen Report volume 1, 1993).

The nucleotide base recognition sequence backbone can be made up ofdifferent groups. Examples of different backbones include asugar-phosphodiester type backbone and a peptide nucleic acid backbone.

Structure I illustrates a sugar-phosphodiester type backbone where thesugar group is a pentofuranosyl group. The sugar groups are joinedtogether by a linkage such as a phosphodiester linkage or other suitablelinkage.

X represents the group joining two sugars. Examples of X include—OP(O)₂O—, —NHP(O)₂O—, —OC(O)₂O—, —OCH₂C(O)₂NH—, —OCH₂C(O)₂O—,—OP(CH₃)(O)O—, —P(S)(O)O— and —OC(O)₂NH—. As with the other examplesprovided herein, other equivalents that are well known in the art orwhich become available can also be used.

Y₁ and Y₂ are independently selected groups. Examples of Y₁ and Y₂include H, OH, C₁-C₄ alkoxy, halogen, and C₁-C₆ alkyl. Preferably, Y₁and Y₂ are independently either H, OH, F, or OCH₃. C₁-C₆ alkyl and C₁-C₄alkoxy, may be or may include groups which are, straight-chain,branched, or cyclic.

Base₁ and Base₂ are independently selected from the group consisting of:adenine, guanine, cytosine, thymine, uracil, or a group that does notinhibit complementary base pairing of an adjacent base to acomplementary nucleic acid. Examples, of groups not inhibitingcomplementary base pairing include smaller size groups such as hydrogen,OH, C₁-C₆ alkyl, and C₁-C₄ alkoxy. Preferably, the nucleotide baserecognition sequence contains about 7 to about 40, more preferably,about 10 to about 30, bases independently selected from the groupconsisting of: adenine, guanine, cytosine, thymine, and uracil.

R₁ and R₂ represent independently selected groups. Examples of R₁ and R₂include additional sugar-phosphodiester type groups, hydrogen, hydroxy,peptide nucleic acid, and molecules not providing sequence informationsuch as abasic “nucleotides”, polysaccharides, polypeptides, peptides,and other non-nucleotide linkages.

Derivatives of Structure I able to be a component of a nucleotide baserecognition sequence are well known in the art and include, for example,molecules having a different type of sugar. For example, a nucleotidebase recognition sequence can have cyclobutyl moieties connected bylinking moieties, where the cyclobutyl moieties have hetereocyclic basesattached thereto. See, e.g., Cook et al., International Application No.PCT/US93/01579, International Publication WO 94/19023 (herebyincorporated by reference herein).

In an embodiment of the present invention, a nucleotide base recognitionmolecule is a polynucleotide or derivative thereof. A “polynucleotide orderivative thereof” is a nucleotide base recognition molecule made up ofstructure I repeating units where X is —OP(O)₂O—; Y₁ and Y₂ areindependently selected groups from the group consisting of H, OH, OCH₃,and F; Base₁ and Base₂ are independently selected from the groupconsisting of: adenine, guanine, cytosine, thymine, uracil, or a groupwhich does not inhibit complementary base pairing of an adjacent base toa complementary nucleic acid; and provided that the molecule containsabout 5 to about 35 bases independently selected from the groupconsisting of: adenine, guanine, cytosine, thymine, and uracil. Theterminal portion of the molecule contains R₁ and R₂ independentlyselected from the group consisting of OH, C₁-C₆ alkyl, and phosphate.

Peptide nucleic acid in a DNA analogue where the deoxyribose phosphatebackbone is replaced by a pseudo peptide backbone. Peptide nucleic acidis described by Hyrup and Nielsen, Bioorganic & Medicinal Chemistry4:5-23, 1996, and Hydig-Hielsen and Godskesen, International ApplicationNumber PCT/DK95/00195, International Publication WO 95/32305, each ofwhich is hereby incorporated by reference herein.

Preferably, the peptide nucleic acid is made up ofN-(2-aminoethyl)glycine units as illustrated in Structure II.

Where R₁, R₂, and Base₁ are as described for Structure I type molecules.

Nucleotide base recognition sequences can be produced using standardtechniques. Publications describing organic synthesis ofoligonucleotides and modified oligonucleotides include Eckstein, F.,Oligonucleotides and Analogues, A Practical Approach, chapters 1-5,1991, that reviews organic synthesis of oligonucleotides; Caruthers etal., Methods In Enzymology vol. 154 p. 287 (1987), that describes aprocedure for organic synthesis of oligonucleotides using standardphosphoramidite solid-phase chemistry; Bhatt, U.S. Pat. No. 5,252,723,that describes a procedure for organic synthesis of modifiedoligonucleotides containing phosphorothioate linkages; and Klem et al.,WO 92/07864, that describes organic synthesis of modifiedoligonucleotides having different linkages including methylphosphonatelinkages. (Each of these references is hereby incorporated by referenceherein.)

Additional references describing techniques that can be used to producedifferent types of nucleotide base recognition sequences include Cook,International Application No. PCT/US92/11339, International PublicationWO 93/13121; Miller et al., International Application No.PCT/US94/00157, International Publication WO 94/15619; McGee et al.,International Application No. PCT/US93/06807, International PublicationWO 94/02051; Cook et al., International Application No. PCT/US93/01579,International Publication WO 94/19023; Hyrup and Nielsen, Bioorganic &Medicinal Chemistry 4:5-23, 1996; and Hydig-Hielsen and Godskesen,International Application Number PCT/DK95/00195, InternationalPublication WO 95/32305. (Each of these references is herebyincorporated by reference herein.)

Labels can be attached to a molecular torch by various means includingcovalent linkages, chelation, and ionic interactions. Preferably, alabel is covalently attached.

Molecular torches present during an amplification protocol preferably donot contain a terminal 3′ OH available for primer extension. Blockinggroups that can inhibit primer extension by a nucleic acid polymerasemay be located at or near the 3′ end of a nucleic acid molecular torch.“At or near” the 3′ end refers to a blocking group present within fivebases of the 3′ terminus. If a blocking group is not placed at the 3′terminus of a nucleic acid molecular torch, it should be sufficientlylarge so as to effect binding of a DNA polymerase to the torch.

Preferably, a nucleic acid molecular torch contains a blocking grouplocated at its 3′ terminus. By attaching a blocking group to a terminal3′ OH, the 3′ OH group is no longer available to accept a nucleosidetriphosphate in a polymerization reaction.

Numerous different chemical groups can be used to block the 3′ end of anucleic acid sequence. Examples of such groups include alkyl groups,non-nucleotide linkers, alkane-diol dideoxynucleotide residues, andcordycepin.

The target binding region should be long enough to bind specifically toa desired target. A bacterial target binding region is preferably atleast about 10 recognition groups, more preferably at least 12recognition groups. A complex target binding region for a multi-cellorganism such as a human, is preferably at least about 16 recognitiongroups, more preferably at least 18 recognition groups.

In an embodiment of the present invention concerned with the targetbinding domain, the target binding domain is made up of about 7 to about40 recognition groups, and 0 to about 4 non-nucleotide monomeric groupseach opposite a recognition group in the target closing domain. Inpreferred embodiments, at least about 8, more preferably at least about10 recognition groups are present; no more than about 30, no more thanabout 25, and no more than about 15 recognition groups are present; andno more than 2, preferably no more than 1, and most preferably 0non-nucleotide monomeric groups are present. Preferably, eachnon-nucleotide monomeric group is an abasic “nucleotide”.

A non-nucleotide monomeric group provides a distance between adjunctgroups containing nucleotide bases which is about the same length as ina nucleic acid. Thus, a non-nucleotide monomeric group joining twonucleotides positions the nucleotides so that they can hydrogen bond tocomplementary nucleotides in a nucleic acid.

In an embodiment of the present invention concerned with the targetclosing domain, the target closing domain is made up of about 7 to about40 recognition groups, and 0 to about 6 non-nucleotide monomeric groupsor mismatches with the target binding domain. In different embodiments,at least about 8, or at least about 10 recognition groups are present;no more than about 30, no more than about 25, and no more than about 15recognition groups are present; and 0, 1, 2, 3, 4, 5 or 6 non-nucleotidemonomeric groups or mismatches with the target binding domain arepresent. Preferably, each non-nucleotide monomeric group is an abasic“nucleotide”. More preferably, mismatches rather than abasic nucleotidesare present.

Preferably, the target binding domain is substantially comprised ofindependently selected 2′-methoxy or 2′-fluoro substitutedribonucleotides, and the target closing domain is substantiallycomprised of independently selected deoxyribonucleotides. “Substantiallycomprised” or “substantially comprises” indicates that the referencedcomponent(s) makes up at least 70%, at least 80%, at least 90%, or 100%of the target opening domain or target closing domain.

The joining region can be produced using techniques well known in theart taking into account the composition of the joining region.Preferably, the joining region contains different members of a bindingset able to bind together, where the target binding domain is joined toone member of the binding set and the target closing domain is joined toanother member of the binding set. A member of a binding set can bind toanother member of the same binding set. Examples of binding sets includesubstantially complementary nucleotide base recognition sequences,antibody/antigen, enzyme/substrate, and biotin/avidin.

Members of a binding set positioned adjacent or near to the targetopening and target closing domains can effect the stability of a targetbinding domain:target closing domain hybrid. Too large an effect canmake it difficult to produce an open torch because the target bindingdomain:target closing domain hybrid may remain intact under a wide rangeof conditions. The affect of a binding set on the stability of a targetbinding domain:target closing domain hybrid can be determined, forexample, by measuring the T_(m) of the hybrid.

Members of binding sets can be covalently linked together using one ormore linkers. Examples of linkers include optionally substituted alkylgroups, polynucleotides and non-nucleotide linkers. Non-nucleotidelinkers include polysaccharides, polypeptides, and abasic “nucleotides”.

Polynucleotides used as linker groups between the target opening andtarget closing domains are preferably designed not to hybridize to thetarget sequence, or other nucleic acids which may be present in thesample. Though some binding to the target may be advantageous, forexample, when strand displacement conditions are used. Preferredpolynucleotide linker groups are poly T, poly A, and mixed poly A-T.Polynucleotide linker groups are preferably 5 to 25 nucleotides.

Molecular torches can include single-stranded regions complementary tothe target sequence that, for example, may be positioned next to thetarget binding domain and may be part of the joining region. Preferably,such single-stranded regions contain no more than about ten nucleotidebase recognition groups complementary to the target sequence. Morepreferably, such single-stranded regions, if present, are no more thanten, five, three, two, or one nucleotide base recognition groupscomplementary to the target sequence.

Linker groups can be positioned between binding set members and thetarget opening and target closing domains to decrease the effect ofbinding set members on the target binding domain:target closing domainhybrid. The placement of linker groups between, for example, a bindingset member and the target binding domain physically separates thebinding set member from the target binding domain thereby decreasing theaffect of the binding set member on the target binding domain:targetclosing domain hybrid.

Additional examples are provided below illustrating different aspectsand embodiments of the present invention. These examples are notintended in any way to limit the disclosed invention.

EXAMPLE 1 Tuning of the Target Binding Domain:Target Closing DomainT_(m)

This example illustrates the use of different molecular torch designfactors to obtain a desired target binding domain:target closing domainhybrid T_(m). The T_(m) of four different molecular torches was adjustedin this example using non-nucleotide, polyethylene glycol (PEG) linkers,a combination of mismatched bases, abasic “nucleotides” (i.e., bulges),and 2′-methoxy substituted ribonucleotides.

The different molecular torches used in this example were constructedfrom four different strands, as shown in FIGS. 6A and 6B. In thesefigures, “F” refers to an EDANS fluorophore, “Q” refers to a DABCYLquencher, and the “ccc” group at the 3′-end of one strand of each torchrefers to a three carbon group which functions as a primer extensionblocking group. The nucleotides of these molecular torches are eitherdeoxyribonucleotides or 2′-methoxy substituted ribonucleotides(indicated with bold/italics). All of the molecular torches used in thisexample contain a joining region composed of a non-nucleotide, 20 Å PEGgroup and a double-stranded, 2′-methoxy substituted ribonucleotideduplex that alone exhibits a very high T_(m) (>90° C.).

As shown in FIG. 6B, Torch 1 is made up of Strands 2 and 3; Torch 2 ismade up of Strands 2 and 4; Torch 3 is made up of Strands 1 and 3; andTorch 4 is made up of Strands 1 and 4. Each of Strands 1-4 (shown inFIG. 6A) includes two nucleotide base recognition sequences separated bya PEG group, where Strand 1 included the nucleotide base recognitionsequences of SEQ ID NO: 1 (5′-cagugcaggn ggaaag-3′) and SEQ ID NO: 2(5′-ggcuggacug cgugcg-3′); Strand 2 included the nucleotide baserecognition sequences SEQ ID NO: 2 and SEQ ID NO: 3 (5′-cagugcaggggaaag-3′); Strand 3 included the nucleotide base recognition sequencesof SEQ ID NO: 4 (5′-cttttccttg ctctg-3′) and SEQ ID NO: 5 (5′-cgcacgcaguccagcc-3′); and Strand 4 included the nucleotide base recognitionsequences of SEQ ID NO: 5 and SEQ ID NO: 6 (5′-ctttnncccc tgcnnactg-3′).

In all four molecular torches, the target binding domain was made up of2′-methoxy substituted ribonucleotides and the target closing domain wasmade up of deoxyribonucleotides. The underlined groups indicatemismatches while “n” denotes abasic bulges.

The stability of different hybrids was determined using a mixturecontaining 500 pmol of each strand added to 350 μl of KEMPS buffer.(KEMPS is made up of 100 mM KCl, 0.1 mM EDTA, 10 mM MgCl₂, 50 mM PIPES(pH 6.85), and 1 mM spermine.) The mixture was heated to 80° C. for 15minutes and then subjected to T_(m) analysis. T_(m) was measuredoptically at 260 nm over a range of 45-95° C. at 0.5° C. min⁻¹ using aBeckman DU-640 melting apparatus.

Table 1 summarizes the stability of the target binding domain:targetclosing domain hybrid in the four molecular torches tested. Table 1 alsohighlights some of the design factors affecting hybrid stability. TABLE1 Melting Temperature Factors Affecting Torch (° C.) Hybrid Stability 176.9 Three mismatched base pairs 2 89.4 Two sets of 2 abasic bulges(“n”) in a deoxy strand 3 64.5 1 abasic bulge (“n”) in a methoxy strand,plus 3 mismatched base pairs 4 72.9 1 abasic bulge (“n”) in methoxy, twosets of 2 abasic bulges (“n”) in a deoxy strandNote:“deoxy” refers to deoxyribonucleotides and “methoxy” refers to2′-methoxy substituted ribonucleotides.

As illustrated in Table 1, the T_(m) of a molecular torch can be tunedusing different factors affecting hybrid stability. Other factorsaffecting hybrid stability, such as those described herein and thosewell known in the art, can also be used to obtain a desired hybrid T_(m)in different solutions.

EXAMPLE 2 Molecular Torch Binding to a Target Sequence

Torch 5, as shown in FIG. 6B, is the same as Torch 1, except that thePEG linker of Strands 2 and 3 was replaced with the deoxyribonucleotidesequences of 5′-tttcttttcttt-3′ and 5′-ttttcttctttc-3′, respectively, sothat the nucleotide base recognition sequences of Torch 5 were SEQ IDNO: 7 (5′-cagugcaggg gaaagtttct tttctttggc uggacugcgu gcg-3′) and SEQ IDNO: 8 (5′-cgcacgcagu ccagcctttt cttctttcct tttccttgct ctg-3′). Torch 5also contained an EDANS fluorophore (“F”) and a DABCYL quencher (“Q”),which were used to detect the presence of a synthetic RNA targetsequence. Torch 5 had a target binding domain (SEQ ID NO: 3) made up of2′-methoxy substituted ribonucleotides (indicated with bold/italics),and a target closing domain (SEQ ID NO: 4) made up ofdeoxyribonucleotides. The target binding domain was perfectlycomplementary to the target sequence but had three mismatches to thetarget closing domain.

Torch 5 was generated by first producing a mixture containing the EDANSand DABCYL strands in KEMPS buffer (as described in Example 1 supra) atpH 6.85. The mixture was heated to 60° C. for 10 minutes and then cooledto room temperature.

Approximately 80 pmol of Torch 5 were incubated with increasing amountsof the RNA target molecule. The sample was heated to 60° C. for 20minutes to open torches and allow for hybridization between the targetbinding domain and the target sequence, and then cooled to roomtemperature to close torches which had not hybridized to the targetsequence. The control sample contained 90 mM of target and was notheated to 60° C. (the sample was maintained at room temperature).

Fluorescence was measured using a Spex Fluorolog-2 spectrophotometer(ISA Jobin Yvon-Spex; Edison, N.J.). The emission wavelength was 495 nm,and the excitation wavelength was 360 nm. Table 2 summarizes the resultsof the experiment. TABLE 2 Incubation Fluorescence Data Temperature RNATarget @ 495 nm (° C.) Level (pmol) (cps) 60 0 11,800 60 30 54,000 60 6089,000 60 90 94,000 Room 90 17,300

The results show that this molecular torch binds approximatelystoichiometrically to the target sequence. And, under the conditionsemployed, the target binding domain is unavailable for binding to thetarget sequence in the absence of heat, thus producing little signaleven in the presence of the target sequence.

EXAMPLE 3 Effect of Different Environments

This example illustrates the use and affect of different solutionenvironments and different torch constructs. The different solutionsused in this example contain different components fortranscription-associated amplification reactions.

Torches 1, 6 and 7 (Torches 1 and 6 are shown in FIG. 6, while Torch 7is not shown) were used in this example. Torch 1 is described in Example1 supra, while Torch 6 contained a 20 Å PEG joining region which joineda 2′-methoxy substituted ribonucleotide target binding domain (SEQ IDNO: 3) (indicated with bold/italics) and a deoxyribonucleotide targetclosing domain (SEQ ID NO: 4). Torch 6 also included a fluoresceinfluorophore (“F”) and a DABCYL quencher (“Q”). Torch 7 was a Torch 6analog, where the target closing domain (SEQ ID NO: 4) was made up ofmodified nucleotides having the phosphodiester linkages replaced withphosphorothioate linkages.

A specified amount of a synthetic RNA target sequence, if any, and 25pmol of molecular torch were mixed together in 100 μl of the solutionwhich was used in creating Conditions A, B, C and D described below.After creating conditions A, B, C and D, each solution was heated to 65°C. for 20 minutes, and then cooled to room temperature for 10 minutes.

Conditions A, B, C and D included reagents selected from the followinggroups of reagents:

-   -   Reagent 1: 40 mM trehalose, 4 mM HEPES, 25 mM Nalc, 0.02 mM        EDTA, 0.04% Triton® X-102, and 0.02 mM zinc acetate, at pH 7.0;    -   Reagent 2: 12.5 mM MgCl₂, 17.5 mM KCl, 0.15 mM zinc acetate, 5%        glycerol, 6.25 mM ATP, 2.5 mM CTP, 6.25 mM GTP, 2.5 mM UTP, 0.2        mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 50 mM Trizma        base, 53 mM trehalose, 100 μM desferoxamine, and 2 mM        spermidine, at pH 8.0;    -   Reagent 3: 18 mM KCl, 4% glycerol, 4 mM HEPES, 0.1 mM EDTA, and        0.0002% phenyl red, at pH 7.0; and    -   Reagent 4: 40 mM trehalose, 4 mM HEPES, 25 mM Nalc, 0.02 mM        EDTA, 0.04% Triton® X-102, 0.02 mM zinc acetate, 18 mM KCl, 4%        glycerol, 4 mM HEPES, 0.1 mM EDTA, and 0.0002% phenyl red, at pH        7.0.

Condition A was made up of 25 μl of Reagent 2, 20 μl of Reagent 4, 50 μlof sample, reverse transcriptase (˜33.4 μg), T7 RNA polymerase (˜540 ng)and primers.

Condition B was made up of 20 μl Reagent 3, 25 μl of Reagent 2, 50 μl ofsample, and primers. Condition C was the same as condition A, butwithout the presence of primers.

Condition D was the same as condition B, but without the presence ofprimers.

Fluorescence was measured using a Spex MicroMax microtiter plate readerand a Fluorolog-2 spectrophotometer using a band pass filter (485 nm) onthe excitation monochromator. Fluorescence was measured using anexcitation wavelength of 491 nm and an emission wavelength of 522 nm.The results are shown in Table 3. TABLE 3 Fluorescence Data RNA Target @522 nm Level (cps) Condition (pmol) Torch 1 Torch 7 Torch 6 A 0 14,00017,000 13,000 (No Torch) 0 13,000 18,000 12,100 5 35,000 25,400 30,00040 68,000 35,100 56,500 B 0 27,000 74,000 27,000 (No Torch) 0 30,00070,000 29,300 5 50,500 78,100 46,700 40 65,000 94,500 67,500 C 0 16,00018,000 14,000 (No Torch) 0 10,000 18,000 13,600 5 39,000 24,100 28,00040 69,000 39,000 54,000 D 0 30,000 82,000 31,000 (No Torch) 0 31,00062,000 35,300 5 54,000 87,100 48,700 40 76,000 106,500 70,500

For each of the different molecular torches examined in the differentenvironments, an increase in signal was observed as the amount of targetsequence increased. The amount of background signal varied dependingupon the torch composition and the environment.

EXAMPLE 4 Detecting Amplified RNA Transcripts

This example illustrates the use of molecular torches present during atranscription-associated amplification procedure (discussed supra) todetect the production of target RNA transcripts.Transcription-associated amplification was performed in the presence ofmolecular torches and, following amplification, the presence of RNAtranscripts was determined with either Torch 6 or a single-stranded,acridinium ester-labeled polynucleotide probe.

For this example, eight separate transcription replicates were generatedin the presence of 20 pmol of Torch 6 and employing conditions specifiedas Condition A in Example 3 supra (except that 1 mM of each dNTP wasemployed instead of the 0.2 mM indicated for Reagent 2) at each of fivedifferent RNA target sequence levels (i.e., 0, 100, 500, 1000 and 5000copies of the target RNA sequence), and for each target sequence level,the eight replicates were pooled into two separate groups of fourreplicates each.

The amplification was carried out at 42° C. Following amplification, 350μl of each pooled reaction solution was heated to 60° C. for 20 minutesto open the molecular torch, thereby permitting hybridization of thetarget binding domain (SEQ ID NO: 3) to the target transcript. Thesample was then cooled to room temperature so that torches which werenot hybridized to target would close.

Torch 6 binding to the target sequence was measured using a SpexMicroMax microtiter plate reader and Spex Fluorolog-2 spectrophotometer.Fluorescence was measured using an excitation wavelength of 491 nm andan emission wavelength of 522 nm.

Acridinium ester-labeled probes perfectly complementary to the targetsequence were used as a control to determine the extent ofamplification. The acridinium ester-labeled probes were employed using ahomogeneous protection assay (“HPA”) format. HPA was carried out on 50μl of each pooled reaction solution using a probe mix containing about3,000,000 total RLUs of acridinium ester-labeled probe and 400 pmol ofcold probe.

HPA formats using acridinium ester-labeled probe to detect targetsequence are described in different references such as Arnold et al.,U.S. Pat. No. 5,283,174, Nelson et al., “Detection Of Acridinium EstersBy Chemiluminescence” in: Nonisotopic DNA Probe Techniques, (Kricka ed.,Academic Press, 1992) pp. 275-311, and Nelson et al., Clin. Chem. Acta194:73-90, 1990, each of which is hereby incorporated by referenceherein.

Tables 4 and 5 provide the results from two different experiments. TABLE4 Experiment 1 Target Copy Fluorescence Data Number HPA Data @ 522 nm(Starting) (RLUs) (cps) 0 7,096 869,000 100 8,711 872,000 9,109 870,000500 29,092 1,377,000 20,910 1,043,000 1000 22,262 1,143,000 7,612860,000 5000 89,389 1,623,000 73,971 1,670,000

TABLE 5 Experiment 2 Target Copy Fluorescence Data Number HPA Data @ 522nm (Starting) (RLUs) (cps) 0 2,879 919,000 4,515 1,080,000 100 11,7891,222,000 9,404 1,159,000 500 14,221 1,379,000 14,641 1,351,000 100019,931 1,697,000 24,052 1,607,000 5000 134,126 3,465,000 3,378 1,045,000

In both of these experiments there was an overall linear relationshipbetween the amount of target RNA transcript detected by HPA using anacridinium ester-labeled probe and the amount of target RNA transcriptsdetected by the molecular torch. In Experiment 1, the sensitivity of theassay was 500 copies of target sequence. In Experiment 2, RNAtranscripts were detected above background from amplification reactionsstarting with 100 copies of target sequence.

EXAMPLE 5 Strand Displacement of Molecular Torch by Target Sequence

This example demonstrates that molecular torches can be designed to bindand detect target sequences under essentially constant environmentalconditions. For this experiment, Torch 8 (see FIG. 7) was designed andtested for its ability to detect an RNA target sequence in solution.Torch 8 was made up of the nucleotide base recognition of SEQ ID NO: 9(5′-cggcugcagg ggaaagaaua gttttttccc ctgcagccg-3′), where the5′-ugcaggggaaagaauag-3′ portion represents the target binding domain,the 5′-tcccctgcagccg-3′ portion represents the target closing domain,the 5′-cggc-3′ portion represents a “clamp” region for binding a portionof the target closing domain sequence, and the 5′-ttttt-3′ portion was adeoxyribonucleotide joining region. A portion of the target bindingdomain (5′-aagaauag-3′) remained unbound to facilitate stranddisplacement of the target closing domain by the target. The targetbinding domain was fully complementary to the target sequence and boththe target binding domain and the clamp region were made up of2′-methoxy substituted ribonucleotides (indicated with bold/italics).The target closing domain was made up of deoxyribonucleotides. Torch 8also included a fluorescein fluorophore (“F”) and a DABCYL quencher(“Q”).

In this experiment, 100 μl of Krammer buffer (20 mM Tris Cl at pH 8.0, 5mM MgCl, and 0.2% Tween®-20) was added to each of 10 microtiter wells ofa white Cliniplate (Labsystems, Inc.; Franklin, Mass.). Increasingconcentrations of the target sequence were added to the 10buffer-containing wells in increasing amounts as indicated in Table 6below, followed by the addition of 30 pmol of Torch 8 to each of the 10wells. The plate was then manually agitated for 10-15 seconds beforecovering the fluid surface of each well with 50 μl of oil, which wasused to limit evaporation and contamination. The plate was maintained atroom temperature for a period of 10 minutes to permit target sequencesufficient time to displace the target closing domain and bind thetarget binding domain. Fluorescence signals from each well were thenmeasured using a Spex MicroMax plate reader nd a Spex Fluorolog-2spectrophotometer, with an emission wavelength of 495 nm and anexcitation wavelength of 525 nm. Table 6 provides the results of thisexperiment. TABLE 6 Fluorescence Data RNA Target @ 525 nm Level (pmol)(cps) 0 4,728 (No Torch) 0 58,460 2.5 99,220 5 149,200 10 290,786 20576,624 40 875,120 50 938,640 100 1,117,912 250 1,190,892

The results of this experiment show that the target sequence was able tostrand invade Torch 8 and bind with the target binding domain at roomtemperature. In addition, the results show that the amount of targetsequence which bound torch in the wells increased with the amount oftarget sequence present in a well, indicating that the torches of thisinvention may also be useful for quantifying the amount of target whichmay be present in a sample.

Other embodiments are within the following claims. Thus, while severalembodiments have been shown and described, various modifications may bemade without departing from the spirit and scope of the presentinvention.

1. A molecular torch for use in detecting the presence of a targetnucleic acid sequence in a sample, said molecular torch comprising: atarget binding domain comprising nucleotide base recognition groups; atarget closing domain comprising nucleotide base recognition groups,wherein said target binding domain is biased toward said targetsequence, such that said target binding domain forms a more stablehybrid with said target sequence than with said target closing domainunder the same assay conditions, and wherein said molecular torch doesnot detectably hybridize under hybridization conditions to said targetsequence when said target binding domain is hybridized to said targetclosing domain; and an unlabeled joining region comprising one or morenon-nucleotide linkers, wherein said joining region joins said targetbinding domain and said target closing domain, and wherein said joiningregion facilitates the formation of a target binding domain:targetclosing domain hybrid in the absence of said target sequence.
 2. Themolecular torch of claim 1, wherein said target binding domain comprises7 to 40 of said nucleotide base recognition groups and 0 to 4non-nucleotide monomeric groups, each said non-nucleotide monomericgroup being opposite one of said nucleotide base recognition groupspresent in said target closing domain.
 3. The molecular torch of claim2, wherein said target closing domain comprises 7 to 40 of saidnucleotide base recognition groups and 0 to 6 non-nucleotide monomericgroups or mismatches with said target binding domain.
 4. The moleculartorch of claim 3, wherein each said non-nucleotide monomeric group is anabasic nucleotide.
 5. The molecular torch of claim 1, wherein at least70% of said nucleotide base recognition groups of said target bindingdomain bind to said nucleotide base recognition groups of said targetclosing domain under said hybridization conditions.
 6. The moleculartorch of claim 1, wherein said target binding domain and said targetclosing domain each comprise a sugar-phosphodiester type linkage andnucleotide base recognition groups able to hydrogen bond to adenine,guanine, cytosine, thymine or uracil joined to said backbone.
 7. Themolecular torch of claim 1, wherein said target binding domain issubstantially comprised of nucleotide base recognition groups which morestably bind to ribonucleotides than to deoxyribonucleotides, and whereinsaid target closing domain is substantially comprised ofdeoxyribonucleotides.
 8. The molecular torch of claim 7, wherein saidtarget binding domain comprises 2′-methoxy or 2′-fluoro substitutedribonucleotides.
 9. The molecular torch of claim 1, wherein at least oneof said non-nucleotide linkers is a polysaccharide or a polypeptide. 10.The molecular torch of claim 1, wherein said target binding domain orsaid target closing domain comprises a label, and wherein said labelproduces a first signal when said target binding domain:target closingdomain hybrid is formed and a second signal when said target bindingdomain:target closing domain hybrid is not formed, said first and secondsignals being distinguishable.
 11. The molecular torch of claim 1,wherein said target binding domain comprises a first label and saidtarget closing domain comprises a second label, wherein said first andsecond labels interact to produce a first signal when said targetbinding domain:target closing domain hybrid is formed and a secondsignal when said target binding domain:target closing domain hybrid isnot formed, said first and second signals being distinguishable.
 12. Themolecular torch of claim 11, wherein said first label is attached to theend of said target binding domain which is not joined to said joiningregion and said second label is attached to the end of said targetclosing domain which is not joined to said joining region.
 13. Themolecular torch of claim 11, wherein said first and second labelscomprise an enzyme/substrate pair, an enzyme/cofactor pair, aluminescent/quencher pair, a fluorophore/quencher pair, aluminescent/adduct pair, a Forrester energy transfer pair or a dye dimerpair.
 14. The molecular torch of claim 1, wherein said molecular torchfurther comprises a blocking group which can inhibit primer extension bya nucleic acid polymerase, and wherein said blocking group is located ator near a 3′ end of said molecular torch.
 15. The molecular torch ofclaim 14, wherein said blocking group is selected from the groupconsisting of an alkyl group, a non-nucleotide linker, an alkane-dioldideoxynucleotide residue and cordycepin.
 16. A method for determiningthe presence of a target nucleic acid sequence in a sample, said methodcomprising the steps of: a) contacting said sample with said moleculartorch of claim 1; b) exposing said sample to denaturing conditions, suchthat said target binding domain and said target closing domain do notform a stable target binding domain:target closing domain hybrid c)exposing said sample to hybridization conditions, such that a targetbinding domain:target closing domain hybrid is formed in the absence ofsaid target sequence and a target binding domain:target sequence hybridis formed in the presence of said target sequence, wherein said targetbinding domain:target closing domain hybrid is more stable than saidtarget binding domain:target closing domain hybrid under saidhybridization conditions; and d) determining whether a target bindingdomain:target sequence hybrid is present in said sample as an indicationof the presence or absence of said target sequence in said sample. 17.The method of claim 16, wherein said target binding domain comprises 7to 40 of said nucleotide base recognition groups and 0 to 4non-nucleotide monomeric groups, each said non-nucleotide monomericgroup being opposite one of said nucleotide base recognition groupspresent in said target closing domain.
 18. The method of claim 17,wherein said target closing domain comprises 7 to 40 of said nucleotidebase recognition groups and 0 to 6 non-nucleotide monomeric groups ormismatches with said target binding domain.
 19. The method of claim 18,wherein each said non-nucleotide monomeric group is an abasicnucleotide.
 20. The method of claim 16, wherein at least 70% of saidnucleotide base recognition groups of said target binding domain bind tosaid nucleotide base recognition groups of said target closing domainunder said hybridization conditions.
 21. The method of claim 16, whereinsaid target binding domain and said target closing domain each comprisea sugar-phosphodiester type linkage and nucleotide base recognitiongroups able to hydrogen bond to adenine, guanine, cytosine, thymine oruracil joined to said backbone.
 22. The method of claim 16, wherein saidtarget binding domain is substantially comprised of nucleotide baserecognition groups which more stably bind to ribonucleotides than todeoxyribonucleotides, and wherein said target closing domain issubstantially comprised of deoxyribonucleotides.
 23. The method of claim22, wherein said target binding domain substantially comprises2′-methoxy or 2′-fluoro substituted ribonucleotides.
 24. The method ofclaim 16, wherein at least one of said non-nucleotide linkers is apolysaccharide or a polypeptide.
 25. The molecular torch of claim 16,wherein said target binding domain or said target closing domaincomprises a label, and wherein said label produces a first signal whensaid target binding domain:target closing domain hybrid is formed and asecond signal when said target binding domain:target closing domainhybrid is not formed, said first and second signals beingdistinguishable.
 26. The molecular torch of claim 16, wherein saidtarget binding domain comprises a first label and said target closingdomain comprises a second label, wherein said first and second labelsinteract to produce a first signal when said target bindingdomain:target closing domain hybrid is formed and a second signal whensaid target binding domain:target closing domain hybrid is not formed,said first and second signals being distinguishable.
 27. The method ofclaim 26, wherein said first label is attached to the end of said targetbinding domain which is not joined to said joining region and saidsecond label is attached to the end of said target closing domain whichis not joined to said joining region.
 28. The method of claim 26,wherein said first and second labels comprise an enzyme/substrate pair,an enzyme/cofactor pair, a luminescent/quencher pair, afluorophore/quencher pair, a luminescent/adduct pair, a Forrester energytransfer pair or a dye dimer pair.
 29. The method of claim 16, whereinsaid molecular torch further comprises a blocking group which caninhibit primer extension by a nucleic acid polymerase, and wherein saidblocking group is located at or near a 3′ end of said molecular torch.30. The method of claim 29, wherein said blocking group is selected fromthe group consisting of an alkyl group, a non-nucleotide linker, analkane-diol dideoxynucleotide residue and cordycepin.
 31. The method ofclaim 16 further comprising separating said molecular torch which hasformed a hybrid with said target sequence from molecular torches presentin said sample which have not formed a hybrid with said target sequence.32. The method of claim 16, wherein said target sequence is the productof an amplification reaction and said molecular torch is added to saidsample prior to initiating said amplification reaction.
 33. A reactionmixture comprising, prior to amplification, said molecular torch ofclaim 1 and a primer for use in generating multiple copies of saidtarget sequence.
 34. The reaction mixture of claim 33, wherein saidtarget binding domain comprises 7 to 40 of said nucleotide baserecognition groups and 0 to 4 non-nucleotide monomeric groups, each saidnon-nucleotide monomeric group being opposite one of said nucleotidebase recognition groups present in said target closing domain.
 35. Thereaction mixture of claim 34, wherein said target closing domaincomprises 7 to 40 of said nucleotide base recognition groups and 0 to 6non-nucleotide monomeric groups or mismatches with said target bindingdomain.
 36. The reaction mixture of claim 35, wherein each saidnon-nucleotide monomeric group is an abasic nucleotide.
 37. The reactionmixture of claim 33, wherein at least 70% of said nucleotide baserecognition groups of said target binding domain bind to said nucleotidebase recognition groups of said target closing domain under saidhybridization conditions.
 38. The reaction mixture of claim 33, whereinsaid target binding domain and said target closing domain each comprisea sugar-phosphodiester type linkage and nucleotide base recognitiongroups able to hydrogen bond to adenine, guanine, cytosine, thymine oruracil joined to said backbone.
 39. The reaction mixture of claim 33,wherein said target binding domain is substantially comprised ofnucleotide base recognition groups which more stably bind toribonucleotides than to deoxyribonucleotides, and wherein said targetclosing domain is substantially comprised of deoxyribonucleotides. 40.The reaction mixture of claim 39, wherein said target binding domaincomprises 2′-methoxy or 2′-fluoro substituted ribonucleotides.
 41. Thereaction mixture of claim 33, wherein at least one of saidnon-nucleotide linkers is a polysaccharide or a polypeptide.
 42. Thereaction mixture of claim 33, wherein said target binding domain or saidtarget closing domain comprises a label, and wherein said label producesa first signal when said target binding domain:target closing domainhybrid is formed and a second signal when said target bindingdomain:target closing domain hybrid is not formed, said first and secondsignals being distinguishable.
 43. The reaction mixture of claim 33,wherein said target binding domain comprises a first label and saidtarget closing domain comprises a second label, wherein said first andsecond labels interact to produce a first signal when said targetbinding domain:target closing domain hybrid is formed and a secondsignal when said target binding domain:target closing domain hybrid isnot formed, said first and second signals being distinguishable.
 44. Thereaction mixture of claim 43, wherein said first label is attached tothe end of said target binding domain which is not joined to saidjoining region and said second label is attached to the end of saidtarget closing domain which is not joined to said joining region. 45.The reaction mixture of claim 43, wherein said first and second labelscomprise an enzyme/substrate pair, an enzyme/cofactor pair, aluminescent/quencher pair, a fluorophore/quencher pair, aluminescent/adduct pair, a Forrester energy transfer pair or a dye dimerpair.
 46. The reaction mixture of claim 33, wherein said molecular torchfurther comprises a blocking group which can inhibit primer extension bya nucleic acid polymerase, and wherein said blocking group is located ator near a 3′ end of said molecular torch.
 47. The reaction mixture ofclaim 46, wherein said blocking group is selected from the groupconsisting of an alkyl group, a non-nucleotide linker, an alkane-dioldideoxynucleotide residue and cordycepin.
 48. The reaction mixture ofclaim 33 comprising a set of primers for use in generating multiplecopies of said target sequence.
 49. The reaction mixture of claim 33,wherein an amplification procedure carried out under essentiallyconstant conditions is used to generate multiple copies of said targetsequence.
 50. The reaction mixture of claim 33, wherein atranscription-associated amplification procedure is used to generatemultiple copies of said target sequence.
 51. The reaction mixture ofclaim 50, wherein said amplification procedure is performed without theaddition of an exogenous RNAse H activity.